1. Field
The invention relates to high-purity protein A recovery from preparations such as supernatants and cell lysates, more particularly, to a process for purification by contacting protein A with a suitable ion exchange material.
2. State of the Art
Several references describing the purification of protein A point the way to conventional purification strategies. One method (Balint, Jr,; U.S. Pat. No. 5,075,423; 1991) involves binding protein A to IgG, or IgG fragments (preferably human IgG), coupled to a suitable support on an anion exchange resin such as DEAE-cellulose. This affinity chromatography process must be followed by ion exchange chromatography to reduce the quantity of contaminants, such as HIV viral envelope antigens and IgG ligand that commonly leaches from the support and elute with the protein A. This process has two main disadvantages:
(i) Although ion exchange chromatography can significantly reduce the amount of antigens and other contaminants leached from the support, this step cannot entirely eliminate these contaminants, making the protein A purified by this process of questionable utility for therapeutic applications;
(ii) Since this process uses both affinity chromatography and ion exchange chromatography, it will tend to be uneconomical for the following reasons:
(a) affinity chromatography resins are expensive and have shorter usable lives than hydrophobic interaction chromatography (HIC) resins or ion exchange resins;
(b) affinity chromatography resins have lower binding capacities than HIC resins or ion exchange resins, requiring the use of larger size equipment, such as tanks, pumps, columns, etc.;
(c) the use of larger size equipment for affinity chromatography requires proportionally greater amounts of processing time, reagents consumed, and generates more waste products which must be discharged or otherwise disposed of;
(d) on a manufacturing scale, the two entirely different process steps referred to above (affinity and ion exchange) would require separate equipment for both process steps.
Another invention process (Love, et al. U.S. Pat. No. 5,314,993; 1994) provides a complicated, multistep method to purify protein A This process has greater utility for therapeutic applications of protein A than that of Balint, Jr., cited above, but is not cost-effective and has the additional disadvantages:
(i) This process requires the protein A preparation to be heated to a temperature of from 50xc2x0 C. to 80xc2x0 C. for 30 seconds which causes xe2x80x9csome contaminating protein to precipitatexe2x80x9d. This procedure is an inefficient purification step since some protein A would certainly be lost by xe2x80x9ctrappingxe2x80x9d and precipitate along with the other proteins thus precipitated. Moreover, using this step to purify protein A on a manufacturing scale (typically 100 liters to 1,000 liters, or more) will require additional capital expenditures for equipment and will consume large amounts of energy;
(ii) Some of the reagents used by the process are environmentally harmful and must be reclaimed or disposed of as hazardous waste.
Protein A is an antibody binding reagent which binds the FC portion of all human antibodies (except IgG3), and some types of antibodies from other animal species such as pig, dog, rabbit, goat, and mouse, among others. Protein A is a component of a wide range of biotechnology and biomedical applications. Protein A may be coupled to supports such as silica, agarose, cellulose, or cross-linked dextran to make affinity chromatography resins to purify antibodies, proteins, peptides, etc. Protein A may be coupled to support, such as agarose, for use as an immunoprecipitant to clarify antibodies from biological solutions. Protein A is commonly used as a component of testing methods, such as agglutination, ELISA, and Western blots, as well as many other well-known applications. Protein A is also used in a growing number of therapeutic applications to bind immunocomplexes and immunoglobulins from blood and serum (Terman, et al.).
Protein A has become so important to biotechnology and biomedicine that xe2x80x9cGenetic Engineering Newsxe2x80x9d on its Free Product Information card, includes it as one of the 30 categories of xe2x80x9cChemicalxe2x80x94Biologicalsxe2x80x9d in which a scientist would be expected to be interested, along with xe2x80x9cOrganic Chemicalsxe2x80x9d, xe2x80x9cFermentation Nutrientsxe2x80x9d and xe2x80x9cCell Culture Mediaxe2x80x9d.
The present invention provides a simple, cost-effective, environmentally benign method of producing high purity, high yield, protein A from a wide variety of protein A preparations. In its simplest embodiment it is essentially a one step process. Using the present invention, a high purity protein A can be obtained by loading a protein A preparation onto a cation exchange column, washing the column with a suitable buffer solution to remove impurities, and eluting the protein A fraction from the column as a single step elution.
Most well-known protein purification schemes, such as those reported in xe2x80x9cPharmacia Separation Newsxe2x80x9d and elsewhere, call for a multistep approach to separate a protein of interest from its impurities. At each step, some impurities are eliminated as a result of properties those impurities share in common, which are not shared by the protein of interest. When the inventors began research aimed at discovering a novel method to purify protein A by means of ion exchange chromatography, they believed a multistep purification scheme was the correct approach. However, during the course of this research, peculiarities were observed regarding the behavior of protein A when bound to strong cation exchange resins, such as Fractogel EMD-SO3, manufactured by EM Separations.
It was unexpectedly discovered that by exploiting certain characteristics inherent in some cation exchange resins, high purity protein A could be separated from a protein A-containing preparation in essentially a single step. The object of the invention process is achieved by carefully controlling the buffer conditions by which protein A, in a protein A-containing preparation, is loaded, washed and eluted. These conditions are not documented in prior art and are not obvious to someone skilled in the art of chromatography.
Protein A purified by the process invention can be economi cally recovered from many types of protein A-containing preparations with high yield and high purity. The simplicity provided by the present invention will reduce the production cost of protein A. Since protein A is the costliest component in most products produced using protein A, reducing the purification cost of protein A should generally benefit the biotechnology and biomedical industries.
The present invention is also more environmentally benign than existing processes. The invention process does not use any reagents which are environmentally harmful. Waste reagents do not need to be reclaimed, or otherwise disposed of as environmentally unsafe. Moreover, the present invention does not use any human- or animal-derived products or by-products, giving the process invention broad utility for human therapeutic applications.
The invention provides a process for the purification of protein A from a wide range of protein A-containing preparations. Purification of high purity protein A by the process invention is achieved:
(i) By removing cells and suspended solids from protein A preparations, such as cell culture supernatants and lysates, by centrifugation or hollow fiber filtration. The source of the protein A may be an extracellular protein A secreted by a natural or recombinant host organism, or from lysates produced to free protein A bond to the cell membranes of various strain of Staphylococcus aureus, or produced intracellularly in recombinant expression organisms, such as Escherichia coli or Bacillus subtilis; 
(ii) Optionally, by using a xe2x80x9cBid Beadxe2x80x9d chromatography resin or fluidized bed technique, cells and suspended solids may not need to be removed from a protein A preparation prior to adsorption;
(iii) Optionally, adsorption on an ion exchange column may be preceded by contacting a protein A preparation with an HIC resin, such as phenyl-HIC, then washing the column with about 100 to 300 mM acetate or citrate buffer, and eluting with distilled or deionized water. (This step is only desirable to save processing time when the salt concentration of a protein A preparation is too high for dilution with distilled or deionized water to be a practical consideration.);
(iv) By adsorbing protein A from a protein A preparation on a suitable cation exchange resin followed by washing and selective elution of the protein A fraction. (This does not generally require ultrafiltration and desalting that is often necessary when adsorption is done on other resins. Instead, preparations can be diluted with distilled or deionized water to a conductivity level that will allow absorption, usually from 1:2 to 1:4, v/v sample to water);
(v) For human therapeutic applications, and other applications where xe2x80x9cultra-purexe2x80x9d protein A is required, the eluent of several separations (about 3 to 5), described in paragraph xe2x80x9civxe2x80x9d above, may be pooled and diluted to about 13 mS with distilled or deionized water. A second separation as described in paragraph xe2x80x9civxe2x80x9d above may then be performed with selective elution of protein A. This separation is done on the same cation exchange column and in the same manner as the separation referred to in paragraph xe2x80x9civxe2x80x9d.
The process invention would not be obvious to someone skilled in the art and provides various advantages over prior art, including certain unexpected advantages compared to the prior art processes. In addition to the advantages provided by the present invention, another advantage is that the process invention does not resort to the use of human or animal components and/or by-products to obtain high purity protein A, making it particularly suitable for the production of human therapeutic products.
Prior art commonly uses IgG affinity resins as an initial capture step to remove protein A from bacterial preparations. In such processes, contamination of the protein A eluent from leached IgG molecules and fragments is unavoidable. Moreover, other human- or animal-derived contaminants may also leach from affinity resins, such as viral antigens that were present with the IgG when it was coupled to the support. These contaminants are reduced by subsequent ion exchange chromatography on an anion exchange resin such as DEAE. Although every precaution is presumably taken to insure that such contaminants are removed during additional downstream processing, not using human- and animal-derived components anywhere in the process offers the greatest probability of producing a contaminant-free product. This is especially important for applications where protein A is used for human therapeutic purposes, such as that described by Bensinger in U.S. Pat. No. 4,614,513; 1986.
An unexpected advantage of the invention process is obtained by means of exploiting certain functional characteristic of cation exchange resins. By so doing, a reduction of processing time, and the amount of reagent needed to purify protein A, is achieved. For many applications, protein A can be suitably separated from a protein A preparation by merely diluting said preparation, adjusting the pH, and eluting the protein A as a substantially pure product. For applications where ultrahigh purity is desired, the eluent-containing protein A fractions from 3 to 6 separations can be pooled and separated a second time using the same chromatography equipment, ion exchange resin, and process as the previous ion exchange separation, except the reagent used during the wash step is reduced to one-fourth that used during the initial separation. This aspect makes the present invention considerably simpler and potentially more economical than prior art.